Recycling of memory B cells between germinal center and lymph node subcapsular sinus supports affinity maturation to antigenic drift

Infection or vaccination leads to the development of germinal centers (GC) where B cells evolve high affinity antigen receptors, eventually producing antibody-forming plasma cells or memory B cells. Here we follow the migratory pathways of B cells emerging from germinal centers (BEM) and find that many BEM cells migrate into the lymph node subcapsular sinus (SCS) guided by sphingosine-1-phosphate (S1P). From the SCS, BEM cells may exit the lymph node to enter distant tissues, while some BEM cells interact with and take up antigen from SCS macrophages, followed by CCL21-guided return towards the GC. Disruption of local CCL21 gradients inhibits the recycling of BEM cells and results in less efficient adaption to antigenic variation. Our findings thus suggest that the recycling of antigen variant-specific BEM cells and transport of antigen back to GC may support affinity maturation to antigenic drift.

Specific comments: 1. For the RNAseq data showing differences between drLN Bmem and disLN Bmem, it would be valuable to know how much of the overall difference is due to the drLN Bmem including cells that are still in cell cycle. How much difference remains once cell cycle signature genes are removed? On that point, how much do the cells in the drLN versus disLN differ in their cell cycle status? Also, is it possible that the differences between drLN and disLN Bmem is reflective of how old the Bmem are rather than these Bmem being separate subsets? As in, the Bmem in the drLN include more newly generated (not yet quiescent) Bmem, whereas those in the disLN were mostly formed longer ago and are likely quiescent. Or perhaps it is that the cells in the disLN have escaped from inflammatory cytokine exposure. These concepts need consideration in the discussion.  (Fig 2G). Which result is more representative? What are the CCR7 surface levels on disLN Bmem by FACS? 3. The effects of ACKR4 deficiency on Bmem cell accumulation in the SCS are striking. However, a limitation with the use of ACKR4 KO mice that needs to be noted is that it cannot establish whether a particular process is Bmem intrinsic or extrinsic. For example, while this may not be any more likely, it is possible that the effects on affinity improvement in Fig 4F involve

altered migration of Tfh cells or of naïve B cells or DCs.
Alternative possibilities such as these need to be considered in the discussion. 4. For the model that CCR7 is guiding Bmem recycling within the drLN, it is inferred that CCR7 ligands are normally available between the SCS and the GC (in the outer follicular mantle). Have the authors attempted to detect CCL21 in this region in WT LNs? CCL21 is not evident here in the WT LN data shown in Ulvmar et al., 2014. If the authors also cannot detect it, perhaps they consider it is below the detection limit, or that CCL19 protein (which is known to be very difficult to detect) is present in this region (although early in situ hybridization data -admittedly of limited sensitivity -did not detect subcapsular signal -Ngo et al., 188, 181, 1998). Perhaps CCL21 entering from peripheral lymphatics is made available on the local subcapsular cells. These possibilities need to be mentioned. 5. The authors note that 'blockade or deletion of CCR6, EBI2 and CXCR3 did not lead to a noticeable change in appearance of Bmem in distLNs'. However, have they examined whether CCR6 contributes to the recycling? Given a report that CCL20 is made by subcapsular lymphatics ( Overall, the discussion section is too short. As well as highlighting the significant implications of the work, more attention needs to be given to the caveats noted above.

Reviewer #2 (Remarks to the Author):
In this study, Zhang and colleagues explored the possibility that some GC-derived memory B cells recycle back to GC in draining lymph nodes and contribute to affinity maturation to antigen drift. While the claim of "recycling" is interesting, it is far from well supported by the data. In fact, the collection/sequence of observations are not necessarily well connected to conclusively show that a subset of GC-derived memory cells come out of the LZ, go into SCS, acquire antigen from macrophages, and then go back into GC to support further affinity maturation. Figure 1, the Cg1-Cre reporter would label not only GC-derived cells but also other activated B cells. The authors need to be careful not to equate GFP+ cells with GC-derived cells. The fact that Blimp1-EGFP+ cells show significant accumulation between the T zone and GC DZ does not necessarily mean that these plasmablasts must have come from GC (B1-8hi can give rise to T-dependent but GC-independent plasma cells) or that some plasmablasts do not come out of GC LZ and then quick travel to the T zone-DZ interface through the follicle. Similarly, the appearance of mKO2+ cells around the GC do not necessarily indicate their GC origin. Because the authors are trying to infer kinetic events from static images, these imprecise correspondence makes it really hard to be convinced of the prescribed event sequences. Figure 1C-D, it seems that some Bcm cells appear 1 day earlier than Bem, and are phenotypically more mature. It is not certain that all Bcm cells in distLN are derived from GC in the reactive LN. It remains possible that some or a majority of those Bcm cells have never gone through the GC reaction.

Response to Reviewer's Comments
Reviewer #1: We thank the reviewers for their positive and constructive reviews, which led to substantial improvements of this manuscript. Data shown in Fig. 1 are from experiments where naïve QM B cells were adoptively transferred one day before immunisation, and the vast majority of memory B cells analysed in Fig 1b, c are newly formed and were generated during the ongoing response. We agree with the reviewer that differences between draining and distant lymph nodes may have developed because the cells have arrived in a non-reactive tissue, or because they have left the GC environment for slightly longer and are "older".

This well performed study reveals a number of interesting insights regarding the migration behavior of newly generated memory B cells (Bmem) in lymph nodes (LNs
As suggested, we have tested the impact of cell cycle associated or inflammatory response genes on the phenotypes. There is some association in the transition from BEM to BCM with cell cycle related genes and little with inflammatory response genes (Fig. I below and new suppl. Fig. S2). Removing either or both these gene sets from our gene expression data did not affect the separation of these population in the principal component analysis.
We would like to point out that we have not followed these "subsets" over time or have tested epigenetic fixation of gene expression patterns. Therefore, we do not know whether the "subsets" we defined in this study represent long term differentiation patterns or transient differentiation stages, as this was not the aim of this study. We have added these considerations to the revised discussion.
Further, while doing this gene set enrichment analysis, we realized that the transition from GC B cells to BEM fits very well with data published by Laidlaw et al. 1 . This is now shown in new suppl. Fig.  S2c. Fig 3b regarding CCR7 (Fig 2G). Which result is more representative? What are the CCR7 surface levels on disLN Bmem by FACS?

With the data in
Apologies for this error. The original dataset contained plasma cells, which had been removed from the dataset, as they did not add valuable information. Unfortunately, an error was made during revision and the plot in Fig. 2h showed the plasma cell instead of the BCM data. This has been corrected. All other data were correct. The complete dataset is now shown in suppl. Fig. S3.

The effects of ACKR4 deficiency on Bmem cell accumulation in the SCS are striking. However, a limitation with the use of ACKR4 KO mice that needs to be noted is that it cannot establish whether a particular process is Bmem intrinsic or extrinsic. For example, while this may not be any more likely, it is possible that the effects on affinity improvement in Fig 4F involve altered migration of Tfh cells or of naïve B cells or DCs. Alternative possibilities such as these need to be considered in the discussion.
This was a concern similarly raised by reviewer #2. We have published that Ack4r4 deficiency affects dendritic cell migration from the SCS in the lymph node parenchyma 2 . We are not aware that Tfh cells do the same journey. In an unpublished study, we performed a large series of experiments to test whether ACKR4 affects affinity maturation and found no changes. Although these data are supposed to be part of a different study, we present some in the new Fig. S11. Caveats are also mentioned in the new discussion. Ulvmar et al., 2014. If the authors also cannot detect it, perhaps they consider it is below the detection limit, or that CCL19 protein (which is known to be very difficult to detect) is present in this region (although early in situ hybridization data -admittedly of limited sensitivity -did not detect subcapsular signal - Ngo et al., 188, 181, 1998). Perhaps CCL21 entering from peripheral lymphatics is made available on the local subcapsular cells. These possibilities need to be mentioned.

For the model that CCR7 is guiding Bmem recycling within the drLN, it is inferred that CCR7 ligands are normally available between the SCS and the GC (in the outer follicular mantle). Have the authors attempted to detect CCL21 in this region in WT LNs? CCL21 is not evident here in the WT LN data shown in
There are a number of studies providing data on CCL19/21 expression in the subcapsular sinus area. Huang et al (Fig. 2) 3 show CCL19 and CCL21 expression in MRC of immunised lymph nodes. Others have not found expression, however, this may be due to issues with sensitivity because nonimmunised lymph nodes were studied 2, 4, 5 .
We have tested CCL21 expression in immunised lymph nodes using immunohistology for RFP or by using CCL21 RFP reporter mice 5 to detect gene expression. In these reactive lymph nodes, CCL21 is expressed under the subcapsular sinus surrounding follicles, particular those containing germinal centres. This is shown in new Fig. 3b and suppl. Fig. S5.
We have no data on CCL19 expression, which, as the reviewer correctly states, is difficult to detect and reporter systems do not exist. This has been added to the discussion.

The authors note that 'blockade or deletion of CCR6, EBI2
and CXCR3 did not lead to a noticeable change in appearance of Bmem in distLNs'. However, have they examined whether CCR6 contributes to the recycling? Given a report that CCL20 is made by subcapsular lymphatics (Zhang et al., eLife 5, e18156, 2016) this needs to at least be discussed. Similarly, is a role of EBI2 in recycling excluded given the prominent expression of Ch25h by marginal reticular cells (Rodda et al., Immunity 48, 1014,  2018)? Also needs to be mentioned.
Thank you for pointing this out. This is all correct. While we were able to test the effects of these different ligands on the appearance of memory B cells in different lymph nodes, it is technically more challenging to follow BEM recycling in presence of absence of these ligands, and we agree that more could be done to fully address this. As suggested, these points have been added to the discussion.

Are the authors able to provide any quantitative information on how many examples of B cells capturing CD169+ material they observed? If this was only seen very rarely, that should be stated.
With the complex architecture, the large and complex shape of the macrophages, and the small amount of material transferred, we were not able to accurately quantify these in vivo observations. We can, however, confirm that these interactions occur regularly. To demonstrate this, we have added a further supplementary videos (suppl. movie 5), showing several occasions where B cells are seen migrating with macrophage material. Nine such interactions are shown, demonstrating that these events are not rare. Fig 4E (assuming these are flow cytometric data) may actually be innate-like lymphocytes that have acquired CD169+ cell membrane material (Gray et al., PlosOne 7, e38238, 2012). Unless microscopy of the isolated cells was done to confirm they are uniformly stained for macrophage markers, the description of the cells should be qualified.

It should be noted that what are described as macrophages in
Thank you for this comment. We are aware that innate-like lymphocytes can acquire CD169. To exclude these cells, all flow cytometry samples were counterstained for CD11b, CD11c, F4/80 to identify macrophages. The full gating scheme for SCS macrophages is now included as suppl. Fig. S9. Apologies for omitting this important information.   Fig S1: the red stromal staining between these samples is very different. At the least some comment about this should be provided in the legend.

Suppl
We note the reviewer's concern with this figure (new Fig. S6). For each condition we presented a 10 µm-thick section of the imaged lymph node where the subcapsular sinus can be visualised clearly. Stromal mTomato signal strength changes significantly at different tissue depths, and varies in different lymph nodes during intravital imaging. As suggested we have clarified this in the figure legend.
4. Overall, the discussion section is too short. As well as highlighting the significant implications of the work, more attention needs to be given to the caveats noted above.
Attention has been given to the caveats.

Reviewer #2 (Remarks to the Author):
In this study, Zhang and colleagues explored the possibility that some GC-derived memory B cells recycle back to GC in draining lymph nodes and contribute to affinity maturation to antigen drift. While the claim of "recycling" is interesting, it is far from well supported by the data. In fact, the collection/sequence of observations are not necessarily well connected to conclusively show that a subset of GC-derived memory cells come out of the LZ, go into SCS, acquire antigen from macrophages, and then go back into GC to support further affinity maturation.
Major concerns: 1. In Figure 1,  We thank the reviewer for this comment. Indeed, our own work shows that immunoglobulin class switching and Cg1 germline expression predates germinal centre formation 6,7,8 . Others have clearly shown that memory B cells can develop independently of germinal centres 9 . At the time of performing this study we had only access to Cg1-Cre and used this as a surrogate marker for GCderived memory B cell generation, as the majority of cells marked by this reporter are GC-derived.
We have repeated some experiments using the GC-specific S1PR2 CreERT2 reporter mouse. New Fig. 5fg and new supplementary movies 8-10 show light sheet microscopy of live lymph nodes with many GC-derived memory B cells between GC and SCS and in the SCS, some in interaction with immune complex. Additionally, we now show data showing that the gene expression profile of BEM is extremely similar to early GC derived memory B cells described by Laidlaw et al 1 (new Fig. S2c).
Regarding the comment on Fig. 1a: We have published extensive evidence that Blimp1-eGFP cells in the area between GC and T zone are GC derived 10 . Fig. 1a is shown mainly as an introduction to explain background and context and explain tissue architecture. Even if some of these plasma cells would have developed outside the GC, this would be irrelevant for the conclusions of the current study. mKO2+ cells: This study is not based on "inferring kinetic events from static images", as we provide several examples of kinetic observations of various events using intravital microscopy (see supplementary movies). Figure 1C-D, it seems that some Bcm cells appear 1 day earlier than Bem, and are phenotypically more mature. It is not certain that all Bcm cells in distLN are derived from GC in the reactive LN. It remains possible that some or a majority of those Bcm cells have never gone through the GC reaction.

In
The reviewer overinterprets data in Fig. 1c. This is probably because BCM numbers for distant LNs on day 4 had not been shown. These were omitted because there is no significant increase in BCM numbers by this day. We have now added these data, showing that BCM appear after the appearance of BEM and GC in the draining lymph node. Fig. 1d shows data from the peak of the response at day 8 after immunisation. The fact that BCM cells appearing in distant LN by day 8 are more phenotypically mature than in the draining LN is not surprising, as they have migrated through lymph and blood by this stage.
We agree that some Cg1Cre cells can be non-GC derived, as discussed above.
3. Live imaging (e.g. Fig 2 and related movies) could help establish some of the event sequences. However, their movies, albeit of good quality, are too short to show GC-derived cells arriving into the SCS or coming out of the SCS. There was no quantitative analysis of their observations in any manner, and it is impossible to know how appropriate their preferred interpretation truly is. I would argue, to establish what they want to say, the authors should consider using light-activated marking of their cells.